MIPC chromatographic apparatus with improved temperature control

ABSTRACT

A liquid chromatography apparatus with stationary and mobile phase temperature controls suitable for polynucleotide separations by MIPC and DMIPC processes. The apparatus includes heater means with a temperature control system; a matched ion polynucleotide chromatography separation column having an inlet end; a coil of capillary tubing having an inlet end and an outlet end. The outlet end of the capillary tubing is connected with the inlet end of the separation column. The inlet end of the capillary tubing comprising means for receiving process liquid, the tubing having a length of from 6 to 400 cm having a linear tubing length of heating means. The separation column and the coil of capillary tubing are enclosed in the heater means. The capillary tubing preferably is PEEK or titanium. The heater means can be an air batch oven. Preferably, it is a heat-conducting block having a first heat transfer surface, a separation column receptacle, and a capillary coil receptacle. A separation column is positioned within the separation column receptacle in heat conducting relationship with an inner wall thereof. A coil of capillary tubing is positioned in the capillary coil receptacle, the outer extremities of the coil being in heat conducting relationship with an inner wall of the capillary coil receptacle. Optimally, the heating means is a Peltier heating and cooling unit in heat conducting relationship with a heat transfer surface of the heating block.

RELATIONSHIP TO COPENDING APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/295,474, filed Apr. 19, 1999, now U.S. Pat. No. 6,103,112 and claimsthe benefit of U.S. Provisional Application No. 60/119,936, filed Feb.12, 1999 and the benefit of U.S. Provisional Application No. 60/099,825,filed Sep. 10, 1998.

This application is related to co-pending, commonly assigned U.S. patentapplication Ser. No. 09/058,580 filed Mar. 10, 1998, Ser. No. 09/058,337filed Mar. 10, 1998, Ser. No. 09/065,913 filed Apr. 24, 1998, Ser. No.09/081,039 filed May 18, 1998, Ser. No. 09/081,040 filed May 18, 1998,and Ser. No. 09/080,547 filed May 18, 1998, the entire disclosures ofall of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention concerns a high pressure chromatographic separation systemdenoted herein as Matched Ion Polynucleotide Chromatography (MIPC) todistinguish it from traditional partitioning-based, reverse phase HPLCsystems. In particular, this invention relates to MIPC chromatographicsystems with improved, high precision column heater systems.

BACKGROUND OF THE INVENTION

Traditional chromatography is a separation process based on partitioningof mixture components between a “stationary phase” and a “mobile phase”.The stationary phase is provided by the surface of solid materials whichcan comprise many different materials in the form of particles orpassageway surfaces of cellulose, silica gel, coated silica gel, polymerbeads, polysaccharides, and the like. These materials can be supportedon solid surfaces such as on glass plates or packed in a column. Themobile phase can be a liquid or a gas in gas chromatography. Thisinvention relates to liquid mobile phases.

The separation principles are generally the same regardless of thematerials used, the form of the materials, or the apparatus used. Thedifferent components of a mixture have different respective degrees ofsolubility in the stationary phase and in the mobile phase. Therefore,as the mobile phase flows over the stationary phase, there is anequilibrium in which the sample components are partitioned between thestationary phase and the mobile phase. As the mobile phase passesthrough the column, the equilibrium is constantly shifted in favor ofthe mobile phase. This occurs because the equilibrium mixture, at anytime, sees fresh mobile phase and partitions into the fresh mobilephase. As the mobile phase is carried down the column, the mobile phasesees fresh stationary phase and partitions into the stationary phase.Eventually, at the end of the column, there is no more stationary phaseand the sample simply leaves the column in the mobile phase.

A separation of mixture components occurs because the mixture componentshave slightly different affinities for the stationary phase and/orsolubilities in the mobile phase, and therefore have different partitionequilibrium values. Therefore, the mixture components pass down thecolumn at different rates.

Since chromatographic separations depend on interactions with thestationary phase, it is known that one way for improving separation isincreasing the surface area of the stationary phase. The bestseparations are obtained when the interactions are low, i.e., thepartitioning coefficient is low and the column is long, providingincreased interactions.

In traditional liquid chromatography, a glass column is packed withstationary phase particles and mobile phase passes through the column,pulled only by gravity. However, when smaller stationary phase particlesare used in the column, the pull of gravity alone is insufficient tocause the mobile phase to flow through the column. Instead, pressuremust be applied. However, glass columns can only withstand about 200psi. Passing a mobile phase through a column packed with 5 micronparticles requires a pressure of about 2000 psi or more to be applied tothe column. 5 to 10 micron particles are standard today. Particlessmaller than 5 microns are used for especially difficult separations orcertain special cases). This process is denoted by the term “highpressure liquid chromatography” or HPLC.

HPLC has enabled the use of a far greater variety of types of particlesused to separate a greater variety of chemical structures than waspossible with large particle gravity columns. The separation principle,however, is still the same.

An HPLC-based ion pairing chromatographic method was recently introducedto effectively separate mixtures of double stranded polynucleotides ingeneral, and DNA in particular, wherein the separations are based onbase pair length (U.S. Pat. No. 5,585,236 to Bonn (1996); Huber, et al.,Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351(1993)). These references and the references contained therein areincorporated herein in their entireties. The term “Matched IonPolynucleotide Chromatography” (MIPC) has been applied to this method bythe Applicants as their understanding of the DNA separation mechanismhas evolved. MIPC separates DNA fragments on the basis of base pairlength and is not limited by the deficiencies associated with gel basedseparation methods.

Matched Ion Polynucleotide Chromatography, as used herein, is defined asa process for separating single and double stranded polynucleotidesusing non-polar separation media, wherein the process uses a counter-ionagent, and an organic solvent to release the polynucleotides from theseparation media. Basic MIPC separations are complete in less than 10minutes, and frequently in less than 5 minutes. For more difficultseparations such as separations using isocratic solvent condition, theseparation time will be longer. Effective isocratic and target zoneelutions require precise control of the separation conditions.

As the use and understanding of MIPC developed, it was discovered thatwhen MIPC analyses were carried out at a partially denaturingtemperature, i.e., a temperature sufficient to denature a heteroduplexat the site of base pair mismatch, homoduplexes could be separated fromheteroduplexes having the same base pair length (U.S. Pat. No.5,795,976; Hayward-Lester, et al., Genome Research 5:494 (1995);Underhill, et al., Proc. Natl. Acad. Sci. USA 93:193 (1996); Doris, etal., DHPLC Workshop, Stanford University, (1997)). These references andthe references contained therein are incorporated herein in theirentireties. Thus, the use of Denaturing HPLC (DHPLC) was applied tomutation detection (Underhill, et al., Genome Research 7:996 (1997);Liu, et al., Nucleic Acid Res., 26;1396 (1998)).

DHPLC can separate heteroduplexes that differ by as little as one basepair. However, separations of homoduplexes and heteroduplexes can bepoorly resolved. Artifacts and impurities can also interfere with theinterpretation of DHPLC separation chromatograms in the sense that itmay be difficult to distinguish between an artifact or impurity and aputative mutation (Underhill, et al., Genome Res. 7:996 (1997)). Thepresence of mutations may even be missed entirely (Liu, et al., NucleicAcid Res. 26:1396 (1998)). The references cited above and the referencescontained therein are incorporated in their entireties herein.

The accuracy, reproducibility, convenience and speed of DNA fragmentseparations and mutation detection assays based on DHPLC have beencompromised in the past because of DHPLC system related problems.Important aspects of DNA separation and mutation detection by HPLC andDHPLC which have not been heretofore addressed include the treatment ofmaterials comprising chromatography system components; the treatment ofmaterials comprising separation media; solvent pre-selection to minimizemethods development time; optimum temperature pre-selection to effectpartial denaturation of a heteroduplex during MIPC; and optimization ofDHPLC for automated high throughput mutation detection screening assays.These factors are essential in order to achieve unambiguous, accurate,reproducible and high throughput DNA separations and mutation detectionresults.

A need exists, therefore, for an HPLC system which can separate DNAfragments based on size differences, and can also separate DNA havingthe same length but differing in base pair sequence (mutations from wildtype), in an accurate, reproducible, reliable manner. Such a systemshould be automated and efficient, should be adaptable to routine highthroughput sample screening applications, and should provide highthroughput sample screening with a minimum of operator attention.

The basic MIPC separation process differs from the traditional HPLCseparation processes in that the separation is not achieved by a seriesof equilibrium separations between the mobile phase and the stationaryphase as the liquids pass through the column. Instead, the sample is fedinto the column using a solvent strength which permits the sample dsDNAto bind to the separation media surface. Strands of a specific base pairlength are irreversibly removed from the stationary phase surface andare carried down the column by a specific solvent concentration. Bypassing an increasing gradient of solvent through the sample,successively larger base pair lengths are removed in succession andpassed through the column. When DNA is released from the stationaryphase, its linear velocity quickly reaches the linear velocity of themobile phase. The basic separation is not column length or stationaryphase area dependent.

An isocratic variation of the MICP process can be applied to separatemixtures of DNA fragments having the same size, where fragments in themixture exhibit differences in the degree of non-polarity.

The application of the Matched Ion Polynucleotide Chromatography (MIPC)under the partially denaturing conditions used for separatingheteroduplexes from homoduplexes in mutation detection is hereafterreferred to as DMIPC.

Separation of double-stranded deoxyribonucleic acids (dsDNA) fragmentsand detection of DNA mutations is of great importance in medicine, inthe physical and social sciences, and in forensic investigations. TheHuman Genome Project is providing an enormous amount of geneticinformation and yielding new information for evaluating the linksbetween mutations and human disorders (Guyer, et al., Proc. Natl. Acad.Sci. USA 92:10841 (1995)). For example, the ultimate source of diseaseis described by genetic code that differs from the wild type (Cotton,TIG 13:43 (1997)). Understanding the genetic basis of disease can be thestarting point for a cure. Similarly, determination of differences ingenetic code can provide powerful and perhaps definitive insights intothe study of evolution and populations (Cooper, et. al., Human Geneticsvol. 69:201 (1985)). Understanding these and other issues related togenetic coding requires the ability to identify anomalies, i.e.,mutations, in a DNA fragment relative to the wild type.

DNA molecules are polymers comprising sub-units called deoxynucleotides.The four deoxynucleotides found in DNA comprise a common cyclic sugar,deoxyribose, which is covalently bonded to any of the four bases,adenine (a purine), guanine (a purine), cytosine (a pyrimidine), andthymine (a pyrimidine), referred to herein as A, G, C, and Trespectively. A phosphate group links a 3′-hydroxyl of onedeoxynucleotide with the 5′-hydroxyl of another deoxynucleotide to forma polymeric chain. In double stranded DNA, two strands are held togetherin a helical structure by hydrogen bonds between what are calledcomplimentary bases. The complimentarity of bases is determined by theirchemical structures. In double stranded DNA, each A pairs with a T andeach G pairs with a C, i.e., a purine pairs with a pyrimidine. Ideally,DNA is replicated in exact copies by DNA polymerases during celldivision in the human body or in other living organisms. DNA strands canalso be replicated in vitro by means of the Polymerase Chain Reaction(PCR).

Sometimes, exact replication fails and an incorrect base pairing occurs.Further replication of the new strand produces double stranded DNAoffspring containing a heritable difference in the base sequence fromthat of the parent. Such heritable changes in base pair sequence arecalled mutations.

As used herein, double stranded DNA is referred to as a duplex. When abase sequence of one strand is entirely complimentary to a base sequenceof the other strand, the duplex is called a homoduplex. When a duplexcontains at least one base pair which is not complimentary, the duplexis called a heteroduplex. A heteroduplex is formed during DNAreplication when an error is made by a DNA polymerase enzyme and anon-complimentary base is added to a polynucleotide chain beingreplicated. Further replications of a heteroduplex will, ideally,produce homoduplexes which are heterozygous, i.e., these homoduplexeswill have an altered sequence compared to the original parent DNAstrand. When the parent DNA has a sequence which predominates in anaturally occurring population, the sequence is generally referred to asa “wild type”.

Many different types of DNA mutations are known. Examples of DNAmutations include, but are not limited to, “point mutation” or “singlebase pair mutations” in which an incorrect base pairing occurs. The mostcommon point mutations comprise “transitions” in which one purine orpyrimidine base is replaced for another and “transversions” wherein apurine is substituted for a pyrimidine (and visa versa). Point mutationsalso comprise mutations in which a base is added or deleted from a DNAchain. Such “insertions” or “deletions” are also known as “frameshiftmutations”. Although they occur with less frequency than pointmutations, larger mutations affecting multiple base pairs can also occurand may be important. A more detailed discussion of mutations can befound in U.S. Pat. No. 5,459,039 to Modrich (1995), and U.S. Pat. No.5,698,400 to Cotton (1997). These references and the referencescontained therein are hereby incorporated by reference in theirentireties.

The sequence of base pairs in DNA is a code for the production ofproteins. In particular, a DNA sequence in the exon portion of a DNAchain codes for a corresponding amino acid sequence in a protein.Therefore, a mutation in a DNA sequence may result in an alteration inthe amino acid sequence of a protein. Such an alteration in the aminoacid sequence may be completely benign or may inactivate a protein oralter its function to be life threatening or fatal. On the other hand,mutations in an intron portion of a DNA chain would not be expected tohave a biological effect since an intron section does not contain codefor protein production. Nevertheless, mutation detection in an intronsection may be important, for example, in a forensic investigation.

Detection of mutations is therefore of great importance in diagnosingdiseases, understanding the origins of disease, and the development ofpotential treatments. Detection of mutations and identification ofsimilarities or differences in DNA samples is also of criticalimportance in increasing the world food supply by developing diseasesresistant and/or higher yielding crop strains, in forensic science, inthe study of evolution and populations, and in scientific research ingeneral (Guyer, et al., Proc. Natl. Acad. Sci. USA 92:10841 (1995);Cotton, TIG 13:43 (1997)).

Alterations in a DNA sequence which are benign or have no negativeconsequences are sometimes called “polymorphisms”. For the purposes ofthis application, all alterations in the DNA sequence, whether they havenegative consequences or not, are defined herein as “mutations”. For thesake of simplicity, the term “mutation” is used herein to mean analteration in the base sequence of a DNA strand compared to a referencestrand (generally, but not necessarily, a wild type). As used herein,the term “mutation” includes the term “polymorphism” or any othersimilar or equivalent term of art.

Prior to this invention, size based analysis of DNA samples wasaccomplished by standard gel electrophoresis (GEP). Capillary gelelectrophoresis (CGE) was also been used to separate and analyzemixtures of DNA fragments having different lengths, e.g., the digestsproduced by restriction enzyme cleavage of DNA samples. However, thesemethods cannot distinguish DNA fragments which have the same base pairlength but have a differing base sequence. This is a serious limitationof GEP.

Mutations in heteroduplex DNA strands under “partially denaturing”conditions can be detected by gel based analytical methods such asdenaturing gradient gel electrophoresis (DGGE) and denaturing gradientgel capillary electrophoresis (DGGC). The term “partially denaturing” isdefined to be the separation of a mismatched base pair (caused bytemperature, pH, solvent, or other factors) in a DNA double strand whileother portions of the double strand remain intact, that is, unseparated.The phenomenon of “partial denaturation” occurs because a heteroduplexwill denature at the site of base pair mismatch at a lower temperaturethan is required to denature the remainder of the strand.

These gel-based techniques are difficult and require highly skilledlaboratory scientists. In addition, each analysis requires a lengthysetup and separation. A denaturing capillary gel electrophoresisanalysis can only be made of relatively small fragments. A separation ofa 90 base pair fragment takes more than 30 minutes. A gradientdenaturing gel runs overnight and requires about a day of set up time.Additional deficiencies of gradient gels are the difficulty of adaptingthese procedures to isolate separated DNA fragments (which requiresspecialized techniques and equipment), and establishing the conditionsrequired for the isolation. The conditions must be experimentallydeveloped for each fragment (Laboratory Methods for the Detection ofMutations and Polymorphisms, ed. G. R. Taylor, CRC Press, 1997). Thelong analysis time of the gel methodology is further exacerbated by thefact that the movement of DNA fragments in a gel is inverselyproportional, in a geometric relationship, to the length of the DNAfragments. Therefore, the analysis time of longer DNA fragments canoften be untenable.

In addition to the deficiencies of denaturing gel methods mentionedabove, these techniques are not always reproducible or accurate sincethe preparation of a gel and running an analysis can be highly variablefrom one operator to another.

Separation of double stranded nucleic acid fragment mixtures by GEP orDGGE produces a linear array of bands, each band in the arrayrepresenting a separated double stranded nucleic acid component of thatmixture. Since many mixtures are typically separated and analyzedsimultaneously in separate lanes on the same gel slab, a parallel seriesof such linear arrays of bands is produced. Bands are often curvedrather than straight, their mobility and shape can change across thewidth of the gel, and lanes and bands can mix with each other. Thesources of such inaccuracies stem from the lack of uniformity andhomogeneity of the gel bed, electroendosmosis, thermal gradient anddiffusion effects, as well as host of other factors. Inaccuracies ofthis sort are well known in the GEP art and can lead to seriousdistortions and inaccuracies in the display of the separation results.In addition, the band display data obtained from GEP separations is notquantitative or accurate because of the uncertainties related to theshape and integrity of the bands. True quantitation of linear band arraydisplays produced by GEP separations cannot be achieved, even when thelinear band arrays are scanned with a detector and the resulting data isintegrated, because the linear band arrays are scanned only across thecenter of the bands. Since the detector only sees a small portion of anygiven band and the bands are not uniform, the results produced by thescanning method are not accurate and can even be misleading.

Methods for visualizing GEP and DGGE separations, such as staining orautoradiography are also cumbersome and time consuming. In addition,separation data is in hard copy form and cannot be electronically storedfor easy retrieval and comparison, nor can it be enhanced to improve thevisualization of close separations.

This MIPC process is temperature sensitive, and precise temperaturecontrol is particularly important in the MIPC separation processesdescribed in co-pending U.S. patent application Ser. No. 09/129,105filed Aug. 4, 1998, for example. Precise temperature control is requiredfor maintaining both mobile and stationary phases at a partiallydenaturing temperature, that is, a temperature at which mismatched DNApresent at the mutation site of a heteroduplex strand will denature butat which the matched DNA will remain bound into the double strand.

In many prior art HPLC applications, the HPLC separation process can berun at room temperature. However, in MIPC and in particular DMIPC,applications are run at elevated temperatures with the temperature ofthe column being thermostatically controlled. Prior art HPLC temperaturecontrols were generally able to set and maintain a temperature withinthe range of ±1° C. and achieve equilibrium temperature within 10minutes. The temperature control did not provide the accuracy orprecision required with the MIPC process. The systems did not providefor establishing and maintaining the column and solution temperatures atthe same value.

With advanced MIPC and DMIPC processes, achieving equilibrium at a newset temperature within 1 minute with a temperature control within therange of ±0.1° C. is needed. In addition, the automated oven controlmust provide the specific oven conditions required for each particularmethod or separation. Maintaining a constant temperature is especiallyimportant in quantitative analysis, since changes in temperature canseriously affect peak-size measurement. These devices usually consist ofhigh-velocity air blowers plus electronically controlled thermostats,with configurations similar to those used in gas chromatographs.Alternatively, LC columns can be jacketed and the temperature controlledby contact heaters or by circulating fluid from a constant-temperaturebath. This latter approach is practical for routine analyses, but doesnot meet the time response requirements, accuracy or precision of theimproved high capacity systems of this invention.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a column heating system inwhich both the temperature of the column and the solutions introduced tothe column are maintained at the same precise and accurate temperatures,features required by the MIPC and DMIPC separation processes carried outtherein.

It is another object of this invention to provide a column heatingsystem in which both the temperature of the column and the solutionsintroduced to the column can be rapidly changed from one temperaturevalue to another without loss of precision or accuracy as required forcertain processes, e.g., empirical temperature titrations to identify anoptimum denaturing MIPC temperature.

In summary, this invention is a liquid chromatography apparatus withstationary and mobile phase temperature control suitable forpolynucleotide separations by MIPC and DMIPC processes. It comprisesheater means having a temperature control system; a matched ionpolynucleotide chromatography separation column having an inlet end; andcapillary tubing having an inlet end and an outlet end. The outlet endof the capillary tubing is connected with the inlet end of theseparation column, and the inlet end of the capillary tubing comprisesmeans for receiving process liquid. The tubing has a fully extendedlength which is preferably from 6 to 400 cm and can be a coil. Theseparation column and the coil of capillary tubing are enclosed in theheater means. The temperature control system is preferably calibrated tothe temperature of liquid flowing into the column.

The capillary tubing is preferably PEEK or titanium. The inlet end ofthe coil of capillary tubing can communicate with a prefilter, theprefilter being optionally enclosed in the heater means.

One embodiment of the liquid chromatography apparatus of this inventionincludes an air bath oven having a recirculating air temperature controlsystem and a matched ion polynucleotide chromatography separation columnhaving an inlet end and an outlet end. It includes a coil of capillarytubing having an inlet end and an outlet end, the outlet end of thecapillary tubing being connected with the inlet end of the separationcolumn, and the inlet end of the capillary tubing comprising means forreceiving process liquid. The tubing preferably has a fully extendedlength of from 6 to 400 cm. The separation column and the coil ofcapillary tubing are enclosed in the air bath oven. Preferably, theinlet end of the coil of capillary tubing communicates with a prefilter,the prefilter being enclosed in air bath oven.

Preferably the apparatus air bath system includes a recirculating airtemperature control system comprising a temperature sensor, a heater anda heater control. The temperature sensor is positioned in the air bathoven in the path of recirculated air and is connected to the heatercontrol. The heater control is connected to the heater, the heater andthe heater control comprising means for regulating the temperature ofrecirculating air in the air bath.

Also this invention includes the process of calibrating the temperaturecontrol by a direct measurement of the temperature of the liquidentering the column, such as an infrared temperature sensor measurementor use of a contact temperature sensor placed on the capillary tubingconnecting the column with the means for receiving process liquid(liquid temperature sensor). A table of corresponding measures of liquidtemperature and air temperature is established. First a systemtemperature is set by a user. Then corresponding air temperature iscalculated from the table. The heater is set to this air temperature.Then the liquid temperature is read by the system control and comparedto the set temperature. If the difference between the set temperatureand the liquid temperature is more than allowed margin, than the heatersettings must be adjusted by increasing or decreasing the heatersettings by the difference between the set temperature and the liquidtemperature. Then the calibration table is adjusted to register theactual set temperature and air and liquid temperatures. This procedureis repeated until the difference between the liquid temperature and theset temperature is within the allowed margin.

Further the temperature sensor can be mounted directly on the coil ofcapillary tubing for more accurate temperature reading.

In another embodiment of this invention, the heater means are comprisedof a heat conducting block having a first primary heat transfer surface,a separation column receptacle, and a capillary coil receptacle. Aseparation column is positioned within the separation column receptaclein heat conducting relationship with an inner wall thereof; and a coilof capillary tubing is positioned in the capillary coil receptacle. Theouter extremities of the coil are in heat conducting relationship withan inner wall of the capillary coil receptacle. The system includes aheater means, the heater means being in heat conducting relationshipwith said first heat transfer surface. The system preferably includes aheater control, the heater control being linked with the heat sensor andthe heater means. The system can include a heat radiator, wherein theheat conducting block has a second heat transfer surface and the heatradiator is in heat conducting relationship with said second heattransfer surface.

Preferably, the heat-conducting block includes a heat sensor receptacleand a heat sensor, the heat sensor being positioned within the heatsensor receptacle in heat conducting relationship with an inner surfacethereof.

The heating means can be a Peltier heating and cooling unit, the Peltierheating and cooling unit being in heat conducting relationship with saidfirst heat transfer surface. The heater control is linked with the heatsensor and Peltier heating and cooling unit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a HPLC DNA analyzer system suitable foruse in separating DNA fragments into fractions based on size.

FIG. 2 is a schematic diagram of a system showing the relationshipbetween the operating components of the DNA analyzer system.

FIG. 3 is a view of a conventional prior art HPLC DNA analyzer columnoven.

FIG. 4 is a front view of the separation compartment of an improved HPLCDNA analyzer column oven according to this invention.

FIG. 5 is a top view of the HPLC DNA analyzer column oven shown in FIG.4.

FIG. 6 is an end view of the compact dual column heater embodiment ofthis invention.

FIG. 7 is a cross-sectional view taken along the line 7—7 in FIG. 6.

FIG. 8 is a schematic view of a Peltier heater.

FIG. 9 is a schematic view of a preferred Peltier heater/coolerembodiment of this invention.

FIG. 10 is a schematic view of a temperature sensor component forcalibration of the temperature controls or for use as the temperaturesensor in the oven shown in FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides an HPLC system which can separate DNA fragmentsbased on size differences by MIPC, and can also separate DNAheteroduplexes of mutant strands paired with wild type strands fromhomoduplexes by DMIPC to identify the presence of mutations. It canachieve these separations in an accurate, reproducible, reliable manner.

Precise temperature control of the separation column and the solutionspassing therethrough is required for optimum DNA fragment separations byMIPC and mutation detection by DMIPC, application of the Matched IonPolynucleotide Chromatography (MIPC) under the partially denaturingconditions used for separating heteroduplexes from homoduplexes inmutation detection.

FIG. 1 is a perspective view of a HPLC DNA analyzer system suitable foruse in separating DNA fragments into fractions based on size. The systemcan comprise stacked components, as shown in this presentation, or itcan be integrated into a single cabinet or housing. The principalcomponents are represented here by the column oven housing 2, whichcontains the separation column, and the detector housing 4 whichcontains a detector which measures a property of the column eluant whichis a function of the concentration of the material being separated. Theautosampler housing 6 contains sample vials or well trays andconventional autosampling components. The pump housing 8 contains thepumps for moving the liquid solvents, reagent solutions, and samplesolutions through the column. The degasser housing 10 houses a solutiondegasser. The control and system monitoring features include a controlinterface 11, a conventional desktop computer with a CPU 12, inputkeyboard 14, and output monitor 16. The system can be connected toconventional printers, networks and auxiliary storage devices (notshown).

FIG. 2 is a schematic diagram of a system showing the relationshipbetween the operating components. Under the directions of the computercontrol 18, eluants 19 are moved by pumps 20 through the autosamplerassembly 22 to the column and column oven 24. The eluants include liquidsolvents, reagent solutions, and sample solutions which are required forthe separation processes. Polynucleotide samples such as DNA fragments,RNA samples and other oligonucleotides are removed from samplecontainers (vials, trays or the like) in the sample source 26 and mixedwith a solvent solution in the autosampler assembly 22. As shown ingreater detail hereinafter, the solutions and the separation column arebrought to a desired controlled temperature in the column oven 24.

The separation column separates polynucleotide components passingtherethrough into size-based fractions, and these fractions are carriedin the eluant solutions passing through the detector 28 and to thefraction collector 30.

The system components, with the exception of the oven, are conventionalHPLC system components which are readily available from domestic andforeign sources. The specialized columns are combined with thesecomponents in the commercial WAVE DNA Fragment Analyzer system availablefrom Transgenomic, Inc. (Omaha, Nebr.).

Details about the MIPC and DMIPC processes, the apparatus and separationcolumns are fully described in U.S. Pat. Nos. 5,585,236, 5,772,889,5,795,976 and co-pending U.S. patent applications Ser. Nos. 09/058,580,09/058,337, 09/129,105, the entire contents of all of which are herebyincorporated by reference.

FIG. 3 is a view of a conventional prior art HPLC DNA analyzer columnoven. Housing 2 contains separation column 32, prefilter 34, andcapillary tube sections 36, 38 and 40. The separation column 32 has aninlet end 42 and an outlet end 44. The inlet end 42 is connected to theprefilter 34 by capillary tube 38. The prefilter 34 communicates withthe autosampler through capillary tube 36. The outlet conduit 40 leadsfrom the outlet end 44, of the column 32 to the analyzer unit. Intraditional HPLC systems, the dead volume represented by the capillarytubing 36, 38, and 40 and the prefilter 34 reduced the separationefficiency, causing peak broadening. Therefore, the lengths of thecapillaries were traditionally minimized.

Air circulating through the housing 2 and passing over the prefilter 34,column 32, and capillary segments 36, 38 and 40 must establish andmaintain these components and the liquid passing through at a selectedtemperature. The housing 2 is separated into a process section and aheater fan section by wall 46. Air exits from the process sectionthrough an exhaust port 48 in the wall 46, drawn by fan 50, passesthrough heater elements 52 and returns to the process section. The exitair temperature is monitored by temperature sensor 54, the sensor 54having a lead connecting with a conventional heater temperaturecontroller 56.

If sufficient time passes to equilibrate the temperature of the systemand system components, the temperature of the tubing, prefilter andcolumn components can be maintained within the range of ±1° C. by thecirculating air. However, air and the system components have a severelylimited opportunity to heat the liquid passing into the column.Temperature uniformity is compromised during operation of the system bythe heat transfer to or from the solutions passing through the capillarytubing and the column.

We have found that the MIPC system is relatively insensitive to thedead-space in the capillary tubing leading from the sample injector andthe separation column. In fact, dead volume up to several millilitersbetween the sample injector and the separation column are acceptable inMIPC systems. The only limitation in dead volume space is its effect onthe total time necessary to form a mobile phase gradient. Unlike HPLC,duality of the separation is not affected by dead space. As aconsequence, the capillary tubing can be lengthened without compromisingthe separation by the column. This feature has permitted the ovenmodifications shown in FIGS. 4 and 5. FIG. 4 is a front view of theprocess compartment of a HPLC DNA analyzer column oven according to thisinvention, and FIG. 5 is a top view of the HPLC DNA analyzer column ovenshown in FIG. 4.

The process compartment in the embodiment shown in FIGS. 4 and 5 isdivided from the heating compartment by wall 58 in which air exhaustport 60 is positioned. A temperature sensor such as thermister 62 ispositioned in the port 60 to monitor the temperature of the air passingthrough the port.

Capillary tubing 64 leads from the sample injector (not shown) to aprefilter 66. Prefilter 66 is an inline filter or guard cartridge suchas described in U.S. Pat. No. 5,772,889. It removes contaminants fromthe incoming liquid. An elongated coil 68 of capillary tubing has aninlet end communication with prefilter 66 for receiving process liquidtherefrom. The elongated coil 68 has an outlet end communicating withthe inlet end 70 of a separation column 72. Separation column 72contains MIPC separation media described in U.S. Pat. No. 5,585,236 andco-pending U.S. patent applications Ser. Nos. 09/058,580, 09/058,337,09/183,123, 09/183,450. Outlet tubing 74 leads from the outlet end 76 ofthe separation column 72 to a detector (not shown).

The coil 68 is a liquid heating coil made of a DNA compatible,multivalent cation free tubing which has the desired heat conductivity.Titanium or PEEK described in U.S. Pat. No. 5,772,889 and applicationSer. No. 09/081,040 are preferred because they can be freed ofmultivalent ion contamination. The length and diameter of tubing used isany length which is sufficient to enable liquid passing therethrough toreach the equilibrium temperature of air in the processing compartment.A length of from 6 to 400 cm and a tubing ID of from 0.15 to 0.4 mm isusually sufficient. Since the length of tubing 68 does not degrade theseparation of components achieved by the system, the length can beselected based on the length required to achieve effective heating ofthe process liquids.

Referring to FIG. 5, the air is heated and recycled in the same manneras described in FIG. 3. Air from the processing compartment 78 passesthrough the opening 60 in wall 58, through a heater/fan system 80 fortemperature adjustment. The adjusted air received by the heatingcompartment 82 recycles back to the processing compartment 78 along thepassageways 84 defined by the spacing between the wall 58 and the outeroven wall 86.

The heating coil in the embodiment shown in FIGS. 4 and 5 provides atemperature accuracy to within the range of ±0.2° C. and reduces thetemperature equilibrium time between temperature settings to below 5minutes for temperature changes of 5° C. and below 2 minutes fortemperature changes for up to 1° C.

FIG. 6 is an end view of a preferred compact dual column heaterembodiment of this invention, and FIG. 7 is a cross-sectional view takenalong the line 7—7 in FIG. 6. This embodiment uses direct metal-to-metalconduction of heat to and from the system components and does not relyon an air bath to achieve temperature changes and accuracy.

This preferred embodiment is shown for a two column system, although itcould be used for a single column, if desired. It comprises heatconducting blocks (88,90) having receptacles sized and shaped to receivethe system components. Filter cavity or prefilter receptacles (92,94)have inner surfaces which are sized to receive prefilters (96,98) andestablish heat transfer contact with the outer surfaces thereof.Separation column receptacles (100,102) have inner surfaces sized toreceive respective separation columns (104,106) and separation columncouplers (108,110) which connect capillary tubing to the respectiveseparation columns. Receptacles (100,102) are sized and shaped toestablish heat transfer contacts between the inner heat transfersurfaces of blocks (88,90) and the separation column components receivedtherein. Capillary coil receptacles 112 (one is shown in FIG. 7) have aninner surface which is shaped to receive a coil of capillary tubing 114(one is shown in FIG. 7) and to establish heat transfer contact with theouter surface thereof. In the embodiment shown in these figures,receptacles (92, 94) and (100, 102) are cylindrical holes withapproximately parallel central axes lying in a common plane. It would bereadily apparent to a person skilled in the art that otherconfigurations are equally suitable and all configurations areconsidered to be within the scope of this invention.

Temperature sensor receptacles (116, 118) are provided in heatconducting blocks (88, 90). Capillary receptacle passageways 112 forreceiving connecting tubing 122 in a heat-conducting relationship arealso provided in the heating-conducting block (88, 90). The capillarycoil receptacles 112 are shown in this figure to be cylindrical cavitieswith their axes perpendicular to the axes of receptacles (92, 94) and(100, 102). Optionally, a conductive metal cylinder (not shown) can bepositioned within the capillary coils in heat conducting contact withthe inner surfaces thereof to increase heat transfer area between themetal block heating assembly and the liquid in the coils.,

A KAPTON resistance heater or other type of heating unit 124 ispositioned between and in heat-conducting contact with surfaces 126 and128 of heating blocks (88, 90) to transfer heat to the heat-conductingblocks. Heat sinks (130, 132) are positioned in heat-conductingrelationship with opposed cooling surfaces (134, 136) of the heatconduct blocks (88, 90) to remove heat therefrom. Cooling fans 138 and140 in a heat removal relationship with the heat sinks 130 and 132 andare activated to accelerate heat removal therefrom.

The heat conducting blocks 88 and 90, and the heat sinks 130 and 132 aremade of a material having high heat conductivity such as aluminum orcopper, although they can be made of other heat-conducting solids suchas ferrous metals or any other solid material having the requisite heatconductivity. Heat pipes can also be used as heat sinks.

The capillary tubing can be made of DNA compatible PEEK or titanium,although titanium is preferred for maximum heat transfer efficiency.With this improved heat transfer, the capillary coil can have a fullyextended length as short as 5 cm although a minimum coil length of 10 cmis preferred. A longer coil of PEEK would be required to achieve thesame heat transfer as titanium capillary tubing.

The system shown in FIGS. 6 and 7 comprises two systems in mirror image.It will be readily apparent that for a single column, half the systemwould be sufficient and is intended to be included within the scope ofthis invention.

The position, alignment and spacing of the receptacles are not acritical feature of this invention. Any alignment and configurationwhich provides a compact and heat-transfer efficient result is intendedto be included within the scope of this invention. Heat transferisolation of the heating and cooling unit from the casing is preferred.

The embodiments of the invention shown in FIGS. 6 and 7 provide acompact heater which is more responsive to heater controls, providesrapid changes from one temperature platform to another, and maintains atemperature accuracy within ±0.5° C. of a set temperature. The heattransfer rate obtained with the metal-to-metal contact between theheating block and the elements being heated is far greater than can beobtained in an air bath system, providing the more rapid response to achanged temperature and greater temperature accuracy. It also allowsprocess liquid temperature adjustment with a shorter capillary tubingcoil.

FIG. 8 is a schematic view of a Peltier heater. With the use ofsemiconductor materials in Peltier elements, relatively high temperaturedifferences can be achieved. Each Peltier element is made of a p- orn-type semiconductor material which is linked to a copper bridge. Inthis view, element 144 is a conventional p-type doped semiconductormaterial such as silicon and element 146 is a conventional n-type dopedsemiconductor material. Elements 148, 150 and 152 are copper bridges.With the negative voltage on the p-doped side and the positive voltageon the n-doped side, copper bridge 148 is cooled and copper bridges 150and 152 are heated. If the voltage is reversed, the copper bridge 148 isheated and the copper bridges 150 and 152 are cooled. Thus with simplevoltage changes, the copper bridge 148 can be a source of both heatingand cooling to rapidly and precisely regulate the temperature of a metalblock in a heat conductive relationship therewith.

FIG. 9 is a schematic view of a preferred Peltier heater/coolerembodiment of this invention. Heating block 154 is in conductive contactwith a Peltier heating element (not shown) for heating or coolingrequired to reach and maintain a desired temperature. Channel 156 is aprefilter receptor having an inner surface 158 in heat conductiverelationship with prefilter 160. Channel 162 is a column and columnguard receptor having an inner surface 163 in heat conductiverelationship with coupler 164 and end nut elements 166 of separationcolumn 168. Capillary tubing 170 communicates with the prefilter 160 andthe sample and solution sources (not shown). Capillary tubing 174 fromthe outlet of the separation column 168 communicates with an analyzer(not shown). Capillary tubing 172 connects the outlet end of theprefilter 160 with the coupler 164, which in turn communicates with theseparation column 168. Capillary tubing 172 is received in alabyrinth-like configuration of channels in the heating block 164 toprovide increased capillary length and surface contact between thecapillary tubing 172 and the heating block 154. The configuration of thelabyrinth can be any configuration which provides an adequate capillarylength and surface contact, including circular loops and capillaryplacement of more than one pass per channel.

The capillary tubing 172 can be PEEK or titanium, titanium beingpreferred because of its high heat conductivity. The heating block 154can be any heat conductive metal. Aluminum or copper are preferredbecause of their higher heat conductivity, although ferrous metals suchas steel can be used.

Temperature sensor 175 is positioned in the center of the block in acorresponding receptor channel.

The Peltier heater is controlled with a conventional temperature andcontrol system (not shown) such as the systems used in Peltierthermocyclers. The temperature accuracy achieved by this Peltier-heatedblock is ±0.5° C.

FIG. 10 is a schematic view of a process liquid temperature sensor.Temperature sensor 176 is mounted on capillary tubing 178 with a layerof thermally conductive grease 180 between the sensor 176 and an outersurface 182 of the tubing 178. The capillary tubing is preferablytitanium. The sensor 176 is further enclosed in an insulation sleeve184. A electrical lead 186 connects the sensor 176 to the temperaturecontrols (not shown). Positioning the temperature sensor 176 directly onthe outer surface of the capillary tubing 178 increases the accuracy oftemperature measurement of the liquid flowing through the tubing A layerof thermally-conductive grease between the sensor 178 and the tubing 178increases the area of thermal contact between the sensor and the tubingand further improves the accuracy of temperature reading.

The temperature sensor of FIG. 10 can also be used for calibrating atemperature sensor mounted in the air bath oven by comparing the datafrom the process liquid temperature sensor to the data from thetemperature sensor located in the air bath oven and calculating thedeviation of the two.

The temperature sensors 62 shown in FIG. 4, the sensors positioned inthe receptors 116 and 118 shown in FIG. 6 and the temperature sensor 175shown in FIG. 9 are optimally calibrated to the temperature of theliquid passing through the capillary tubing and separation column.

This invention is further described in the following specific butnon-limiting examples. In these examples, procedures described in thepast tense in the examples below have been carried out in thelaboratory. Procedures described in the present tense have not beencarried out in the laboratory and are constructively reduced to practicewith the filing of this application.

EXAMPLE 1

A WAVE brand DNA fragment separation system from Transgenomic Inc. wascombined with an oven unit produced from a Model PTC200 M J Researchthermocycler. The thermocycler was modified to contain a DNASep™ columnand preheat lines (150 cm×0.33 mm ID) made of PEEK tubing. The preheattubing was interwound between the PCR tube wells (i.e., physicallyplaced around the wells themselves and in thermal contact with the96-well heating block) and then was connected to the column placed in acavity machined out of the thermocycler. The oven response was high withapproximately 10 seconds required to reach a set temperature. It tookabout 2 minutes for the fluid to reach the set temperature. Thisresponse was much faster than the air bath oven of FIGS. 4 and 5. Theoven was cooled and heated with a Peltier unit, so that increases anddecreases in temperature were effected rapidly.

While the foregoing has presented specific embodiments of the presentinvention, it is to be understood that these embodiments have beenpresented by way of example only. It is expected that others willperceive and practice variations which, though differing from theforegoing, do not depart from the spirit and scope of the invention asdescribed and claimed herein.

The invention claimed is:
 1. A liquid chromatography apparatus forpolynucleotide separations by MIPC and DMIPC processes comprising:heater means comprising a heat conducting block having a first heattransfer surface, a separation column receptacle, and a capillary coilreceptacle; a separation column positioned within the separation columnreceptacle in heat conducting relationship with an inner wall thereof;and a coil of DNA compatible capillary tubing positioned in thecapillary coil receptacle, the outer extremities of the coil being inheat conducting relationship with an inner wall of the capillary coilreceptacle.
 2. A liquid chromatography apparatus of claim 1 including aheater in heat conducting relationship with said first heat transfersurface.
 3. A liquid chromatography apparatus of claim 1 including aheater control, the heater control being linked with the heat sensor andthe heater means.
 4. A liquid chromatography apparatus of claim 1including a heat radiator, wherein the heat conducting block has asecond heat transfer surface and the heat radiator is in heat conductingrelationship with said second heat transfer surface.
 5. A liquidchromatography apparatus of claim 1 wherein the heat conducting blockincludes a heat sensor receptacle and a heat sensor, the heat sensorbeing positioned within the heat sensor receptacle in a heat conductingrelationship with an inner surface thereof.
 6. A liquid chromatographyapparatus of claim 1 including a Peltier heating and cooling unit, thePeltier heating and cooling unit being in heat conducting relationshipwith said first heat transfer surface.
 7. A liquid chromatographyapparatus of claim 6 including a heater control, the heater controlbeing linked with a heat sensor and the Peltier heating and coolingunit.